The primary objectives of the proposed research are the isolation and characterization of centromeric regions of yeast chromosomes (Saccharomyces cerevisiae). The isolation of such regions will be accomplished by the use of recombinant DNA techniques to clone centromere-linked genes and the subsequent use of overlap hybridization procedures to obtain long stretches of DNA flanking these cloned genes as a series of overlapping recombinant plasmids and phage. These studies are designed to elaborate the molecular organization of the DNA sequences surrounding the centromere on individual chromosomes and to compare the organization of this region between different chromosomes. The general organization of repetitive DNA sequences, as well as other non-transcribed sequences and genes coding for transfer RNA and messenger RNA species will be determined and detailed characterization of anumber of these elements will be carried out by renaturation kinetics analyses, electron microscopy, transcription studies and DNA sequencing. It is anticipated that the DNA sequence corresponding to the centromere itself can be isolated on a recombinant molecule and analyzed by DNA sequencing techniques, and that the interactions of this stretch of DNA with proteins involved in the mitotic spindle, such as tubulin and microtubule-associated proteins will be examined. These experiments should provide valuable basic information which will contribute to an understanding of the organization and expression of a simple eukaryotic genome.